CN113846021A - Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder - Google Patents
Liquid culture method of sparassis crispa, freeze-dried powder and application of freeze-dried powder Download PDFInfo
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- CN113846021A CN113846021A CN202111038438.5A CN202111038438A CN113846021A CN 113846021 A CN113846021 A CN 113846021A CN 202111038438 A CN202111038438 A CN 202111038438A CN 113846021 A CN113846021 A CN 113846021A
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- sparassis crispa
- liquid culture
- glucan
- residue
- culture
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Classifications
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Abstract
The invention belongs to the technical field of sparassis crispa cultivation, and particularly relates to a liquid culture method of sparassis crispa, sparassis crispa dry powder and application. The liquid culture method of sparassis crispa provided by the invention adopts sparassis crispa MH-3 (preservation number FERM P-17221) as an initial culture strain, and can culture in a short time to obtain the sparassis crispa liquid culture solution containing high-concentration (more than 60 weight percent) beta-1, 3-D-glucan (6 bifurcations). Compared with sporophore products, the sparassis crispa liquid culture solution of mycelium provided by the invention can be used for preparing sparassis crispa dry powder containing high-concentration (more than 60 wt%) beta-1, 3-D-glucan (6 ramification) stably, and the sparassis crispa dry powder can be applied to preparing various functional foods, medicines, beverages, health care products and cosmetics raw materials in a liquidization mode, so that the uniformity and the content stability of the raw materials are possible.
Description
Technical Field
The invention belongs to the technical field of Sparassis crispa industrial development, and particularly relates to a liquid culture method of Sparassis crispa, Sparassis crispa freeze-dried powder, a method for extracting beta-1, 3-D-glucan from Sparassis crispa freeze-dried powder, and application of Sparassis crispa freeze-dried powder.
Background
Sparassis crispa, also known as Sparassis crispa, a scientific name of Latin, is of the genus Sparassis, the family Sparassis, the genus Sparassis, the order Aphyllophorales. Sparassis crispa is a very rare and rare fungus used both as food and medicine, grows on conifers such as larch, and is called "Wangu king" and "fantastic miracle" in Japan because of its extremely high ability to activate immunity. Sparassis crispa contains a large amount of protein, carbohydrate, various vitamins, various amino acids, dietary fiber, ash, minerals, etc. Wherein, the sparassis crispa contains a large amount of beta-glucan which is a bioactive substance and has multiple functions of immunoregulation, anti-tumor, anti-inflammation, antivirus, antioxidation, antiradiation, blood sugar reduction, blood fat reduction, liver protection, hematopoietic function improvement and the like. The inventors have conducted studies on the biological activity of Sparassis crispa, and have found that Sparassis crispa extract contains beta-1, 3-D-glucan (6 diverged) in a solvent extract thereof, and have obtained a patent on Sparassis crispa extract (patent document: JP 4183326B 2).
Sparassis crispa grows slowly and is difficult to carry out industrial artificial cultivation. Patent document two (JP3509736B2) discloses that Sparassis crispa MH-3 (deposit number NITE FERM BP-17221) is obtained by culturing fruiting body (mushroom) artificial cultivation technique based on wood bacterial bed. The second technique of patent document is to produce a bed for artificial cultivation mainly from sawdust and artificially cultivate Sparassis crispa in a cultivation period of about 3 to 4 months, so that Sparassis crispa can be cultivated in a short time at low cost, but it cannot be guaranteed that the produced Sparassis crispa contains a physiologically active ingredient beta-1, 3-D-glucan (6 divergences). The content of beta-1, 3-D-glucan (6 diverged) in sparassis crispa products provided by the prior art is greatly different due to different strains and culture methods.
Hyphae (hyphal. hyphale) are filamentous structures that make up the main part of a fungus. The fungus body composed of hyphae is called mycelium (mycelium pl. The growth is obtained by the spread of individual hyphae to the trunk, and by the enzymes in the hyphae, the decomposition and absorption of the nutrients of the trunk. The structure of the fruit body, which grows from the bark and grows as a mushroom, appears in appearance. Each hypha is a unit that can sustain life independently, and even if hypha is divided, the hypha can continue to grow. The main component of the hyphal cell wall is polysaccharide, most of which is chitin, including beta-glucan and chitosan. Sparassis crispa (mushroom) is composed of hyphae aggregates. The Sparassis crispa (mushroom) is a brown humus mushroom different from other mushrooms, grows from the tip part (under soil) of the root, but does not grow from the trunk, and has very slow growth of hypha and fruiting body, which is symbiotic with mold.
Since the growth of hyphae is slow in the artificial solid culture technology, liquid culture of various hyphae is researched and produced in a trial way all the time, but no mature technology for liquid culture of the Sparassis crispa mycelium exists so far, and no research report on extraction and analysis of various active ingredients contained in the Sparassis crispa mycelium exists. Therefore, there is a need to develop a technology for extracting stable high-concentration and high-purity beta-1, 3-D-glucan (6 ramifications) from Sparassis crispa liquid culture method and a liquid culture medium thereof to fill the blank of the technical field.
Disclosure of Invention
In view of the above-mentioned technical problems of the background art, there is a need for a liquid culture method of Sparassis crispa, which combines the advantages of short culture period of Sparassis crispa and stable high concentration of β -1, 3-D-glucan (6 bifurcations) in Sparassis crispa obtained by culture, and can maintain stable high concentration and high purity of β -1, 3-D-glucan (6 bifurcations) in the cold alkali extract obtained from the liquid culture medium of Sparassis crispa, and a lyophilized powder of Sparassis crispa and its application.
To achieve the above object, in a first aspect of the present invention, the inventors provide a liquid culture method of sparassis crispa, comprising:
a first culturing step: inoculating a Sparassis crispa strain into an agar culture medium containing a first nutrient component for culturing to obtain a first culture strain, wherein the Sparassis crispa strain is Sparassis crispa MH-3 with the preservation number of FERM BP-17221, and the agar culture medium containing the first nutrient component contains: soaking banana powder, cerevisiae Fermentum, peptone, Mel powder, wheat flour, calcium, magnesium chloride, powdered agar and needle-leaved tree in 40-80 deg.C hot water to obtain extractive solution with pH of 6.0-7.0;
a second culturing step: inoculating the first culture strain into an agar culture medium containing a second nutrient component, and performing monospore culture for 7-15 days in an ultraviolet environment to obtain a second culture strain;
a third culturing step: inoculating the second culture strain into a 150ml Erlenmeyer flask liquid culture medium containing a third nutrient component, and performing shake culture for 7-15d to obtain a third liquid culture solution;
the nutrient components added to the agar culture medium of the second nutrient component and the liquid culture medium of the 150ml Erlenmeyer flask of the third nutrient component comprise: 1L of culture solution water, 1.5-20g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate and 1g of peptone;
a fourth culturing step: and (3) diluting the third liquid culture solution to 10 times, transplanting the third liquid culture solution into a 150mL Erlenmeyer flask, and continuing culturing for 10-15d to obtain a fourth liquid culture solution.
Sparassis crispa strain MH-3 used in the present invention was deposited at 17.2.1999 in the independent administration organization for product assessment and technology infrastructure (NITE) with deposit number FERM BP-17221 and the patent microorganism preservation center NPMD, wherein 100g of Sparassis crispa dry powder obtained by culturing the strain contains 60g or more of beta-1, 3-D-glucan (6 diverged).
The agar medium containing the first nutrient component is added with various nutrient components to promote the growth of the sparassis crispa strain. Specifically, for example, banana powder, brewer's yeast, peptone, honey powder, wheat flour, calcium, magnesium chloride, powdered agar, needle-leaved tree hot water extract (for example, including but not limited to extract after needle-leaved tree larch and red pine are soaked with hot water at 40-80 ℃), and other nutrient substances such as hypha active nutrients of wheat, wheat bran, barley, corn, etc. The agar medium is preferably slant agar medium to enlarge the area of the hyphal culture medium. In addition, the pH of the agar medium was maintained in the range of 6.0-7.0. For example, a first culture strain is obtained by storing a sterilized agar medium in a test tube, inoculating Sparassis crispa MH-3, and culturing.
Various nutrients were added to the agar medium to promote growth of the first cultured strain: 1L of culture solution water, 1.5-20g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate and 1g of peptone. Culturing in ultraviolet at 18-23 deg.C to activate. The culture period is preferably 7-15 days, and the second cultured strain is obtained.
And a third culturing step of inoculating the second cultured strain into a 150ml Erlenmeyer flask liquid culture medium containing nutrient components, and inoculating the second cultured strain in 5 plates of culture dishes into 5 Erlenmeyer flasks respectively for culturing. Various nutrients were added to the liquid medium in the Erlenmeyer flask to promote the growth of mycelia. Specifically, the liquid medium containing the third nutrient component contains, for example: 1L of culture solution water, 1.5-20g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate and 1g of peptone. Culturing at 18-23 deg.C, preferably for 7-15 days.
In the fourth culturing step, 300mL of 2 liquid culture medium in 5 flasks of 150mL can be inoculated into 20 flasks each containing 150mL of liquid culture medium at a 10% inoculum size. The culture time is preferably 10-15 days, and a fourth liquid culture solution is obtained.
In a second aspect of the present invention, the inventors provide a lyophilizate of Sparassis crispa, which is obtained by centrifuging the fourth liquid culture medium of the first aspect of the present invention to remove water, and freeze-drying. In a specific example, 3000ml of the fourth liquid culture medium obtained in the fourth culturing step was centrifuged to remove water, and lyophilized to obtain a lyophilized powder of Sparassis crispa.
Preferably, the sparassis crispa dry powder contains 60 wt% or more of β -1, 3-D-glucan (6 ramifications).
In a third aspect of the present invention, the inventors provide a method for extracting β -1, 3-D-glucan (6 ramifications) from a sparassis crispa lyophilized powder as described in the second aspect of the present invention, comprising the steps of:
defatting 10.8g of the lyophilized powder of Sparassis crispa with ethanol at room temperature for 2 days to obtain a first extract and a first residue;
adding 100 deg.C hot water into the first residue, and autoclaving for 2 hr to obtain a second extract and a second residue;
adding 500ml of the treatment solution of NaOH/urea into the second residue, standing at 4 ℃ for 2 days, stirring, precipitating with ethanol, dehydrating, and drying to obtain beta-1, 3-D-glucan and a third residue.
Preferably, the NaOH/urea treatment solution contains 10% of NaOH and 5% of urea in percentage by mass.
Preferably, the method for extracting beta-1, 3-D-glucan from the Sparassis crispa lyophilized powder according to the second aspect of the present invention comprises the steps of:
carrying out degreasing treatment on 10.8g of the sparassis crispa freeze-dried powder by using ethanol at room temperature for 2 days to obtain a first extract and a first residue;
adding 500ml of 100 deg.C hot water into the first residue, and automatically autoclaving for 2h to obtain a second extract and a second residue;
and adding 500ml of an NaOH/urea treatment solution into the second residue, standing at 4 ℃ for 2D, stirring at 3000rpm for 10min, precipitating with ethanol, dehydrating, and drying to obtain beta-1, 3-D-glucan.
In a fourth aspect of the present invention, the inventors provide a beta-1, 3-D-glucan extracted by the method according to the third aspect of the present invention, wherein the content of glucose in the beta-1, 3-D-glucan is higher than 90%.
In a fifth aspect of the present invention, the inventors provide an application of the lyophilized powder of Sparassis crispa in the second aspect of the present invention in the preparation of functional foods, beverages, and health products. The sparassis crispa freeze-dried powder prepared by the sparassis crispa liquid culture method can be mixed with sugar or sugar alcohols, meat or fish extracts, proteins, peptides, amino acids, dietary fibers, vitamins, starches, dextrins, oils, alcohols, salts, seasonings, spices, preservatives, sweeteners, pigments, quality improving agents, spices and the like, and can be prepared into liquid, solid and powdery functional foods, beverages and health-care products.
In a sixth aspect of the present invention, the inventors provide a use of the lyophilized powder of Sparassis crispa according to the second aspect of the present invention in the preparation of a medicament. Beta-1, 3-D-glucan (6 bifurcate) has various effects of resisting tumor, infection and virus, resisting oxidation, preventing radiation, reducing blood fat, protecting liver, reducing blood sugar, activating immunity and the like. Therefore, the composition can be prepared into various forms and various purposes of medicines such as tablets, powder, pills, liquid, injection and the like by adding various auxiliary materials.
In a seventh aspect of the present invention, the inventors provide a use of the lyophilized powder of Sparassis crispa according to the second aspect of the present invention in the preparation of cosmetics. The beta-1, 3-D-glucan (6-branched) has whitening effect, wrinkle improving effect, and moisturizing effect. Therefore, these effects can be imparted to cosmetics by adding the lyophilized powder of Sparassis crispa obtained by the present invention to the cosmetics. Can be mixed with water (purified water, hot spring water, deep water, etc.), oil agent, surfactant, metal soap, humectant, gelling agent, alcohols, water-soluble polymer, powder, pH regulator, skin membrane forming agent, resin, ultraviolet ray protectant, inclusion compound, antibacterial agent, perfume, deodorant, salt, algefacient, animal and microorganism-derived extract, plant extract, blood activity promoter, proliferation agent, lipid leakage inhibitor, active oxygen scavenger, cell activator, keratolytic agent, enzyme, hormone, vitamin, etc. to make into cosmetic, daily use product, etc.
The solvent of the cosmetic is water-soluble or water-insoluble, preferably water-soluble. The water-soluble solvent is water or a water-soluble solvent mainly containing water. For example, water, alkaline water to which an alkali or other salt-based substance is added, an aqueous solution to which an organic solvent such as ethanol is added, or an acidic aqueous solution to which an acid or an acidic substance is added. As the water-insoluble solvent, a polar organic solvent such as dimethyl sulfite (DMSO) is most commonly used. Dimethylformamide (DMF) may be preferred. In the case of extraction with water as a solvent, hot water is more preferable. The temperature of the water is preferably 80-125 ℃.
Different from the prior art, the technical scheme at least has the following beneficial effects:
(1) the liquid culture method of sparassis crispa provided by the invention adopts sparassis crispa MH-3 (preservation number FERM P-17221) as an initial culture strain, and can culture in a short time to obtain the sparassis crispa liquid culture solution containing high-concentration (more than 60 weight percent) beta-1, 3-D-glucan (6 bifurcations).
(2) Compared with the solid culture method of fruit body, the liquid culture of the mycelium is not influenced by the temperature, the meteorological environment, the difference of the raw materials of the fungal bed, the mould and the air pollution, does not need large mushroom production facilities and production machines, and reduces the water and electricity cost and the cost of maintenance and repair expenses.
(3) Compared with a sporophore product, the method for preparing the sparassis crispa freeze-dried powder by the mycelium sparassis crispa liquid culture solution can stably obtain the sparassis crispa freeze-dried powder containing high-concentration (more than 60 weight percent) beta-1, 3-D-glucan (6 ramifications).
(4) The Sparassis crispa lyophilized powder contains high concentration (more than 60 wt%) of beta-1, 3-D-glucan (6 ramification), and can be applied to the liquefaction of raw materials for preparing various functional foods, medicines, beverages, health care products and cosmetics by utilizing the physiological activity of the Sparassis crispa lyophilized powder, so that the uniformity of the raw materials and the stability of the content of the raw materials are possible.
(5) The cold alkali extract of the sparassis crispa freeze-dried powder contains high-purity beta-1, 3-D-glucan (6 bifurcation) and the convenience and short period of the operation of the whole production process make the planned production of the raw material of the beta-1, 3-D-glucan (6 bifurcation) very easy to realize.
Drawings
FIG. 1 is a collection of images of Sparassis crispa MH-3 according to an embodiment;
FIG. 2 is a diagram illustrating the culture of Sparassis crispa in the UV environment in the second culturing step according to the embodiment;
FIG. 3 is a drawing showing a second cultured strain cultured in a liquid medium in the third culturing step according to the embodiment;
fig. 4 is a picture of a lyophilized powder of Sparassis crispa according to the embodiment.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application.
In the examples of the present invention, the microorganism, Sparassis crispa MH-3 strain, which belongs to the genus Sparassis and has been deposited at 17.1999 in NiBH (now named independent administrative agency for human product evaluation technology basic agency, the patent microorganism Collection (NITE-IPOD)) under the accession number FERM P-17221, and international deposited at 5.12.2019 under the Budapest treaty, and given the International patent microorganism deposit number FERM BP-17221. However, the culture method of Sparassis crispa provided by the invention is not limited by the Sparassis crispa MH-3 provided by the invention, and can be applied to the culture of other Sparassis crispa strains.
Test materials, reagents and the like used in the following specific examples are commercially available unless otherwise specified.
Example 1
Referring to fig. 1-3, the liquid culture method of sparassis crispa according to the present embodiment includes the following steps:
a first culturing step: the method comprises the steps of inoculating a Sparassis crispa strain, which is Sparassis crispa MH-3 (see figure 1) deposited with NITE of independent administration organization for product assessment technology infrastructure organization NITE and having a deposit number of NITE FERM BP-17221, into an agar medium containing a first nutrient component, and culturing to obtain a first culture strain, wherein the Sparassis crispa strain is added with various nutrient components to promote growth of the Sparassis crispa strain. The nutritional ingredients added in this example comprise: banana powder, beer yeast, peptone, honey powder, wheat flour, calcium, magnesium chloride, powdered agar and needle-leaved tree hot water extract, in this example, the extract of needle-leaved tree larch and red pine after being soaked in 50 deg.C hot water is selected, and other nutrient substances such as hypha active nutrient of wheat, wheat bran, barley, corn, etc. The agar medium is preferably slant agar medium to enlarge the area of the hyphal culture medium. Further, the pH of the agar medium was 6.0. In this example, the sterilized agar medium was stored in a test tube, and Sparassis crispa MH-3 was inoculated and cultured to obtain a first culture strain.
Please refer to fig. 2 for the second cultivation step: inoculating the first cultured strain into agar medium containing second nutrient component, and culturing under ultraviolet for 7d to obtain second cultured strain. Various nutrients were added to the agar medium to promote growth of the first cultured strain: 1L of culture solution water, 1.5g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of monopotassium phosphate and 1g of peptone. Culturing in ultraviolet at 18 deg.C to activate. The culture period was 7 days, and a second cultured strain was obtained.
Referring to the third culturing step of FIG. 3, the second cultured strain was inoculated into a 150ml Erlenmeyer flask liquid medium containing nutrients, and the second cultured strain in 5 plates was inoculated into 5 Erlenmeyer flasks, respectively, for culturing. Various nutrients were added to the liquid medium in the Erlenmeyer flask to promote the growth of mycelia. The liquid medium containing the third nutrient of this example contains: 1L of culture solution water, 1.5g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of monopotassium phosphate and 1g of peptone. The culture was carried out at a temperature of 18 ℃ for 8 days.
In the fourth culturing step, 300mL of 2 liquid culture medium in 5 flasks of 150mL were inoculated into 20 flasks each containing 150mL of liquid culture medium at an inoculum size of 10%. The culture time was 15 days, and a fourth liquid culture medium was obtained.
3000ml of the fourth liquid culture medium obtained in the fourth culturing step of this example was centrifuged to remove water, freeze-dried, and pulverized into a desired powder size to obtain a lyophilized powder of Sparassis crispa as shown in FIG. 4.
Through detection, the sparassis crispa freeze-dried powder contains beta-1, 3-D-glucan (6 ramifications) more than or equal to 60%.
Example 2
The difference from example 1 is that the hot water extract of conifer is obtained by soaking the extract of the needle-leaved pine alone in 80 ℃ hot water, the pH of the agar medium in the first culturing step is 7.0, the starch in the second culturing step is 10g, glucose is 20g, magnesium sulfate is 1g, potassium dihydrogen phosphate is 2g, and peptone is 1g, and the conifer is cultured in a single spore culture at 20 ℃ under ultraviolet environment for 10 days, and the liquid medium containing the third nutrient in the third culturing step contains 10g of starch and is cultured at 20 ℃ for 10 days.
3000ml of the fourth liquid culture medium obtained in the fourth culturing step of this example was centrifuged to remove water, and freeze-dried to give a lyophilized powder of Sparassis crispa of this example. Through detection, the sparassis crispa freeze-dried powder contains beta-1, 3-D-glucan (6 ramifications) more than or equal to 60%.
Example 3
The difference from example 1 is that the hot water extract of conifer is obtained by soaking the extract of the needle-leaved pine alone in 80 ℃ hot water, the pH of the agar medium in the first culturing step is 6.5, the starch in the second culturing step is 20g, glucose is 20g, magnesium sulfate is 1g, potassium dihydrogen phosphate is 2g, and peptone is 1g, and the conifer is cultured in a single spore culture at 23 ℃ under ultraviolet environment for 15 days, and the liquid medium containing the third nutrient in the third culturing step contains 20g of starch and is cultured at 20 ℃ for 15 days.
3000ml of the fourth liquid culture medium obtained in the fourth culturing step of this example was centrifuged to remove water, and freeze-dried to give a lyophilized powder of Sparassis crispa of this example. Through detection, the sparassis crispa freeze-dried powder contains beta-1, 3-D-glucan (6 ramifications) more than or equal to 60%.
Example 4
The difference from example 1 is that the hot water extract of conifer is obtained by soaking the extract of the needle-leaved pine alone in 40 ℃ hot water, the pH of the agar medium in the first culturing step is 6.0, the starch 15g, glucose 20g, magnesium sulfate 1g, potassium dihydrogen phosphate 2g and peptone 1g are cultured in the second culturing step in the presence of ultraviolet light at 22 ℃ for 15 days, and the liquid medium containing the third nutrient component in the third culturing step contains starch 15g and is cultured at 20 ℃ for 15 days.
3000ml of the fourth liquid culture medium obtained in the fourth culturing step of this example was centrifuged to remove water, and freeze-dried to give a lyophilized powder of Sparassis crispa of this example. Through detection, the sparassis crispa freeze-dried powder contains beta-1, 3-D-glucan (6 ramifications) more than or equal to 60%.
Example 5
The difference from example 1 is that the hot water extract of conifer is obtained by soaking the extract of the needle-leaved pine alone in 50 ℃ hot water, the pH of the agar medium in the first culturing step is 6.5, the starch in the second culturing step is 5g, glucose is 20g, magnesium sulfate is 1g, potassium dihydrogen phosphate is 2g, and peptone is 1g, and the conifer is cultured in a liquid medium containing 20g of starch at 20 ℃ and under ultraviolet environment for 7 days, and the third culturing step is carried out in a liquid medium containing 20g of starch at 20 ℃ and under 20 ℃ for 10 days.
3000ml of the fourth liquid culture medium obtained in the fourth culturing step of this example was centrifuged to remove water, and freeze-dried to give a lyophilized powder of Sparassis crispa of this example. Through detection, the sparassis crispa freeze-dried powder contains beta-1, 3-D-glucan (6 ramifications) more than or equal to 60%.
Example 6
A method for extracting beta-1, 3-D-glucan from the lyophilized powder of Sparassis crispa obtained in examples 1-5, comprising the steps of:
10.8g of the lyophilized powder of Sparassis crispa obtained in examples 1-5 was mixed and soaked in ethanol for two days for degreasing treatment to obtain a first extract and a first residue, the first residue was separated, 500mL of hot water at 100 ℃ was added, and autoclaving treatment was performed automatically for 2 hours to separate a second extract and a second residue.
Adding 500mL of 10% NaOH and 5% urea treatment solution into the second residue, standing at 4 deg.C for 2d, stirring at 3000rpm for 10min, precipitating with ethanol, dehydrating, and drying to obtain cold alkali extract and third residue.
Sugar content detection is carried out on the cold alkali extract, and the glucose content in the beta-1, 3-D-glucan of the cold alkali extract is more than or equal to 90 percent, namely the purity value of the beta-1, 3-D-glucan in the cold alkali extract provided by the method reaches more than 90 percent.
Leukocytes are composed of 5 cells, increased neutrophils, such as bacterial infections and inflammation; increased lymphocytes, such as viral infections and lymphomas; increased eosinophils, such as increased allergies and parasites; basophils are increased, such as leukemia and the like, and monocytes are increased in the recovery phase of infection.
When immune function is enhanced, the balance of 5 kinds of leukocytes is active. The safety and effectiveness of beta-1, 3-D-glucan (6 divergences) in the sparassis crispa freeze-dried powder and the extract thereof obtained from sparassis crispa MH-3 liquid culture solution are confirmed in animal experiments, and the sparassis crispa freeze-dried powder and the extract thereof have the effects of increasing and repairing all five kinds of reduced leukocytes in a balanced manner, so that the autoimmune system reaches the optimal balanced state.
Beta-1, 3-D-glucan (6 diverged) (from mushroom) injection is widely used in clinic, and has strong immune enhancement effect and anticancer effect. Beta-1, 3-D-glucan (6 divergences) of Sparassis crispa MH-3 is of a pure structure type, and the middle island expert team discovers the effectiveness of oral administration of the beta-1, 3-D-glucan (6 divergences) of mice for the first time in the world and publishes various immune enhancement effects. The application of the sparassis crispa dry powder provided in examples 1 to 5 in the preparation of functional foods, beverages and health-care products. The sparassis crispa liquid culture method of sparassis crispa prepares sparassis crispa dry powder, can be mixed with sugar or sugar alcohols, meat or fish extracts, proteins, peptides, amino acids, dietary fibers, vitamins, starches, dextrins, oils, alcohols, salts, seasonings, spices, preservatives, sweeteners, pigments, quality improving agents, spices and the like, and can be prepared into liquid, solid and powdery functional foods, beverages and health care products.
The use of the dry powder of Sparassis crispa provided in examples 1-5 for the preparation of a medicament. Beta-1, 3-D-glucan (6 bifurcate) has various effects of resisting tumor, infection and virus, resisting oxidation, preventing radiation, reducing blood fat, protecting liver, reducing blood sugar, activating immunity and the like. Therefore, the composition can be prepared into various forms and various purposes of medicines such as tablets, powder, pills, liquid, injection and the like by adding various auxiliary materials.
Use of the dry powder of Sparassis crispa provided in examples 1-5 for the preparation of a cosmetic. The beta-1, 3-D-glucan (6-branched) has whitening effect, wrinkle improving effect, and moisturizing effect. Therefore, these effects can be imparted to cosmetics by adding the lyophilized powder of Sparassis crispa obtained by the present invention to the cosmetics. Can be mixed with water (purified water, hot spring water, deep water, etc.), oil agent, surfactant, metal soap, humectant, gelling agent, alcohols, water-soluble polymer, powder, pH regulator, skin membrane forming agent, resin, ultraviolet ray protectant, inclusion compound, antibacterial agent, perfume, deodorant, salt, algefacient, animal and microorganism-derived extract, plant extract, blood activity promoter, proliferation agent, lipid leakage inhibitor, active oxygen scavenger, cell activator, keratolytic agent, enzyme, hormone, vitamin, etc. to make into cosmetic, daily use product, etc.
The solvent of the cosmetic is water-soluble or water-insoluble, preferably water-soluble. The water-soluble solvent is water or a water-soluble solvent mainly containing water. For example, water, alkaline water to which an alkali or other salt-based substance is added, an aqueous solution to which an organic solvent such as ethanol is added, or an acidic aqueous solution to which an acid or an acidic substance is added. As the water-insoluble solvent, a polar organic solvent such as dimethyl sulfite (DMSO) is most commonly used. Dimethylformamide (DMF) may be preferred. In the case of extraction with water as a solvent, hot water is more preferable. The temperature of the water is preferably 80-125 ℃.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (10)
1. A liquid culture method of sparassis crispa is characterized by comprising the following steps:
a first culturing step: inoculating a Sparassis crispa strain into an agar culture medium containing a first nutrient component for culturing to obtain a first culture strain, wherein the Sparassis crispa strain is Sparassis crispa MH-3 with the preservation number of FERM BP-17221, and the agar culture medium containing the first nutrient component contains: soaking banana powder, cerevisiae Fermentum, peptone, Mel powder, wheat flour, calcium, magnesium chloride, powdered agar and needle-leaved tree in 40-80 deg.C hot water to obtain extractive solution with pH of 6.0-7.0;
a second culturing step: inoculating the first culture strain into an agar culture medium containing a second nutrient component, and performing monospore culture for 7-15 days under ultraviolet rays to obtain a second culture strain;
a third culturing step: inoculating the second culture strain into a 150ml Erlenmeyer flask liquid culture medium containing a third nutrient component, and performing shake culture for 7-15d to obtain a third liquid culture solution;
the nutrient components added to the agar culture medium of the second nutrient component and the liquid culture medium of the 150ml Erlenmeyer flask of the third nutrient component comprise: 1L of culture solution water, 1.5-20g of starch, 20g of glucose, 1g of magnesium sulfate, 2g of potassium dihydrogen phosphate and 1g of peptone;
a fourth culturing step: and (3) diluting the third liquid culture solution to 10 times, transplanting the third liquid culture solution into a 150mL Erlenmeyer flask, and continuing culturing for 10-15d to obtain a fourth liquid culture solution.
2. A lyophilized powder of Sparassis crispa, which is obtained by centrifuging the fourth liquid culture medium of claim 1 to remove water, and lyophilizing.
3. The Sparassis crispa lyophilized powder of claim 2, wherein the Sparassis crispa dried powder contains 60 wt% or more of β -1, 3-D-glucan.
4. A method for extracting β -1, 3-D-glucan from sparassis crispa lyophilized powder according to claim 2, comprising the steps of:
defatting the lyophilized powder of Sparassis crispa with ethanol to obtain a first extract and a first residue;
adding 100 deg.C hot water into the first residue, and autoclaving for 2 hr to obtain a second extract and a second residue;
adding 500ml of the treatment solution of NaOH/urea into the second residue, standing at 4 ℃ for 2 days, stirring, precipitating with ethanol, dehydrating, and drying to obtain beta-1, 3-D-glucan and a third residue.
5. The method of claim 4, wherein the NaOH/urea treatment solution comprises 10% NaOH and 5% urea by mass.
6. The method of claim 5, comprising the steps of:
carrying out degreasing treatment on 10.8g of the sparassis crispa freeze-dried powder by using ethanol at room temperature for 2 days to obtain a first extract and a first residue;
adding 500ml of 100 deg.C hot water into the first residue, and automatically autoclaving for 2h to obtain a second extract and a second residue;
and adding 500ml of an NaOH/urea treatment solution into the second residue, standing at 4 ℃ for 2D, stirring at 3000rpm for 10min, precipitating with ethanol, dehydrating, and drying to obtain beta-1, 3-D-glucan.
7. The beta-1, 3-D-glucan extracted according to the method of claim 4 or 5, wherein the content of glucose in the beta-1, 3-D-glucan is higher than 90%.
8. Use of the lyophilized powder of Sparassis crispa according to claim 2 or 3 in the preparation of functional food, beverage, and health product.
9. Use of a lyophilized powder of Sparassis crispa according to claim 2 or 3 for the preparation of a medicament.
10. Use of a lyophilized powder of Sparassis crispa according to claim 2 or 3 for the preparation of a cosmetic product.
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